With the long-term goal of understanding how synaptic signaling regulates neuronal connectivity and contributes to neurological disease, we propose to use Drosophila as a model system for determining the molecular mechanisms by which spontaneous neurotransmitter release is regulated and how it may regulate synaptic connectivity. Characterization of neuronal communication at synapses has largely focused on action potential-triggered synaptic vesicle fusion, with spontaneous miniature potentials (minis) largely thought to represent background noise. However, we have recently discovered that spontaneous release at synapses is regulated independently of evoked release by complexin, a synapse-specific SNARE complex binding protein, and may modulate synaptic growth. Mutations in Drosophila complexin lead to a dramatic increase in spontaneous fusion at synapses that is independent of extracellular calcium or action potentials, and cause a profound overgrowth of synaptic boutons. Complexin is also a target for PKA-dependent phosphorylation, suggesting that mini frequency can be independently regulated at synapses in a PKA-dependent manner. Regulation of spontaneous fusion by a complexin fusion clamp provides a new avenue for information transfer at neuronal synapses independent of evoked synaptic potentials. In addition, alterations in complexin levels in humans have been implicated in a host of neurological diseases ranging from schizophrenia to Parkinson's and Huntington's Disease, suggesting complexin dysfunction and abnormal rates of spontaneous release may contribute to several human neuropathologies. Here, we propose to take advantage of the genetic tools available in Drosophila to determine how complexin modulates spontaneous release and if its function can be regulated by neuronal activity and the PKA pathway. The completion of these experiments will allow us to propose future studies to define the biological role of minis and their mode of regulation in the brain, potentially providing a fundamental advance in our understanding of the biology of the synapse and neuronal communication mediated by minis - the type of high risk/high reward experiments ideal for R21-level support. PUBLIC HEALTH RELEVANCE: Characterization of synaptic communication has largely focused on action potential-triggered synaptic vesicle fusion, with spontaneous miniature potentials (minis) largely thought to represent background noise. Our recent work suggests that minis can function in a signaling role at the synapse, and are regulated by the neuron specific protein complexin. We will explore how the complexin-SNARE machinery regulates spontaneous release at synapses and contributes to synaptic growth and plasticity. Defining the role of minis in the biology of the synapse would be an important advance in our understanding of brain function and set the stage for further understanding of how complexin dysregulation contributes to neurological disease.